Summary

In this study, we elucidate the roles of the winged-helix transcription
factor Foxa2 in ventral CNS development in zebrafish. Through cloning of
monorail (mol), which we find encodes the transcription
factor Foxa2, and phenotypic analysis of mol-/- embryos,
we show that floorplate is induced in the absence of Foxa2 function but fails
to further differentiate. In mol-/- mutants, expression of
Foxa and Hh family genes is not maintained in floorplate cells and lateral
expansion of the floorplate fails to occur. Our results suggest that this is
due to defects both in the regulation of Hh activity in medial floorplate
cells as well as cell-autonomous requirements for Foxa2 in the prospective
laterally positioned floorplate cells themselves.

Foxa2 is also required for induction and/or patterning of several distinct
cell types in the ventral CNS. Serotonergic neurones of the raphé
nucleus and the trochlear motor nucleus are absent in
mol-/- embryos, and oculomotor and facial motoneurones
ectopically occupy ventral CNS midline positions in the midbrain and
hindbrain. There is also a severe reduction of prospective oligodendrocytes in
the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two
likely Hh pathway target genes are ectopically expressed in more dorsal
regions of the midbrain and hindbrain ventricular neuroepithelium, raising the
possibility that Foxa2 activity may normally be required to limit the range of
action of secreted Hh proteins.

Introduction

The floorplate is a band of non-neuronal cells in the ventral neural tube
that acts as an organising centre to pattern the ventral CNS
(Strähle et al., 2004).
At early stages, the floorplate is a source of secreted Hedgehog (Hh) proteins
that establish a gradient within the ventral neural tube
(Jacob and Briscoe, 2003;
Ruiz i Altaba et al., 2003).
As a consequence, different classes of neurone and glial cells are established
at discrete dorsoventral (DV) positions depending upon the level of Hh
activity to which precursor cells are exposed. Hh signals also have important
roles in mediating cell proliferation and perhaps survival of at least some
CNS cells (Thibert et al.,
2003; Ruiz i Altaba et al.,
2002a; Ruiz i Altaba et al.,
2002b). At later developmental stages, the floorplate mediates
axon guidance through the expression of a variety of secreted guidance cues
that direct both DV and longitudinal trajectories of axons
(Kaprielian et al., 2001;
Charron et al., 2003).

The floorplate of both tetrapods and teleosts has distinct lateral and
medial subdivisions that are distinguished by expression of different
repertoires of genes and may have different embryonic origins
(Odenthal et al., 2000;
Charrier et al., 2002;
Strähle et al., 2004). In
zebrafish, the two subdivisions of the floorplate are named medial floorplate
(MFP) and lateral floorplate (LFP)
(Odenthal et al., 2000).
Expression of genes including sonic hedgehog (shh),
tiggywinkle hedgehog (twhh) and the transcription factor
encoding foxa1 (previously forkhead7) are restricted to the
MFP, whereas foxa2 (axial, hnf3b) and foxa
(forkhead4, fkh4, pintallavis) are expressed in both MFP and LFP
cells (Odenthal et al., 2000;
Odenthal and Nüsslein-Volhard,
1998).

In mouse, one of the targets of Hh signalling within prospective floorplate
tissue is Foxa2, which encodes a winged-helix transcription factor.
Foxa2 expression is absent from the ventral CNS of mouse mutants
lacking activity of the Hh transcriptional effector Gli2, and exogenous Shh
can induce ectopic Foxa2 expression (reviewed by
Strähle et al., 2004).
Reciprocally, Foxa2 consensus binding sites are present within the enhancers
of the mouse and zebrafish shh genes
(Chang et al., 1997;
Müller et al., 1999;
Epstein et al., 1999;
Jeong and Epstein, 2003) and
ectopic Foxa2 activity can induce shh expression
(Hynes et al., 1995). These
studies suggest that Hh and Foxa2 act in a positive feedback loop, thereby
explaining the homeogenetic inductive properties of floorplate tissue. It has
proven difficult to test this model so far, as Foxa2 knockout mice
fail to form a node and the consequent absence of axial tissues has precluded
analysis of the direct role of this gene in floorplate development
(Ang and Rossant, 1994;
Weinstein et al., 1994).

In zebrafish, LFP-specific expression of foxa2 is absent in Hh
pathway mutants, suggesting dependence on Hh activity for transcription in
this region (Odenthal et al.,
2000; Schauerte et al.,
1998). However, within the MFP, it appears that foxa2 is
induced and functions downstream of the Nodal pathway. For example,
cell-autonomous activation of Nodal signalling leads to induction of
foxa2 expression (Müller et
al., 2000), and exogenous Foxa2 can rescue medial floorplate gene
expression in Nodal pathway mutants
(Rastegar et al., 2002). These
and other observations have led to the suggestion that during floorplate
induction in fish, Foxa2 functions downstream of Nodal signals
(Rastegar et al., 2002;
Strähle et al., 2004),
analogous to the role proposed for Foxa2 acting downstream of Hh activity in
mammals.

In this study, we elucidate the role of Foxa2 in patterning the ventral CNS
through phenotypic analysis of mol-/- mutant zebrafish
embryos that carry mutations in the foxa2 gene.
mol-/- embryos show a fully penetrant and fully expressed
phenotype in which a floorplate forms but fails to expand or differentiate.
Our results suggest that Nodal-dependent induction of floorplate occurs in the
absence of Foxa2 activity but that subsequent steps in floorplate development
depend heavily upon functional Foxa2. We also show that absence of Foxa2
function leads to defects in the development of oligodendrocytes, the
serotonergic raphé nucleus and several cranial motor nuclei.

Materials and methods

Mutant/gene nomenclature

smu is the slow muscle omitted mutation, which disrupts
the smoothened gene; syu is the sonic-you mutation,
which disrupts the sonic hedgehog gene.

Fish stocks

Embryos were obtained by natural spawnings from wild-type and
monorail (mol)tv53a
(Brand et al., 1996),
monorail (mol)st20, smoothened
(smu)b641
(Barresi et al., 2000;
Varga et al., 2001) and
sonic-you (syu)tq252
(Schauerte et al., 1998) fish.
mol-/- fish expressing the isl1:GFP transgene were
obtained from parents generated by crossing a heterozygous
moltv53a carrier with a homozygous Tg(isl1:GFP) fish
(Higashijima et al., 2000).
mol-/-;syu-/- double mutant fish were generated
by crossing fish heterozygous for both moltv53a and
syutq252. The moltv53a and
molst20 alleles have very similar phenotypes and
moltv53a mutants were used for most of the phenotypic
characterisation.

Pharmacological blockade of Hh signalling

For inhibition of Hh pathway signalling, embryos were incubated in 100μ
M cyclopamine (diluted in fish water from 20 mM stock in ethanol) between
4 hours and the time of fixation for analysis of floorplate differentiation or
between 36 hours and 48 hours for experiments analysing ectopic ntn1
and nk2.2a expression (see Results). Treatment was terminated by
three washes in fish water followed by fixation. To exclude the possibility
that observed defects were due to ethanol treatment, control embryos were
incubated in an equivalent concentration of ethanol without cyclopamine.
Furthermore, cyclopamine treatment gave rise to phenotypes well established as
being due to abrogation of Hh activity, such as partial cyclopia and U-shaped
somites.

Microinjection and rescue experiments

foxa2 morpholino (5′-CCTCCATTTTGACAGCACCGAGCAT-3′,
Gene-Tools) was diluted in 1 × Danieau buffer [58 mM NaCl, 0.7 mM KCl,
0.4 mM MgSO4, 0.6 mM Ca (NCO3)2, 5.0 mM HEPES
pH 7.6] and stored at -20°C. Synthetic mRNAs were synthesised using the
SP6 mMessage mMachine transcription kit (Ambion) according to the
manufacturer's protocol. Embryos were injected at the single cell stage with
either 4 ng/μl morpholino, or with between 50 ng/μl and 100 ng/μl
mRNA. Rescue experiments were performed using mRNA transcribed from a
pCS2MT:foxa2-ER plasmid (see
Rastegar et al., 2002).
Protein activity was initiated at between 50% epiboly and 70% epiboly by
treatment with 10-7 M βEstradiol (Sigma). Control experiments
were performed using mRNA transcribed from a pCS2MT:hER plasmid
containing no foxa2 sequence. shh RNA was injected at the
single cell stage at a concentration of 50 ng/μl and injected embryos were
left to develop for up to three days before fixation.

Linkage analysis, genetic mapping, cloning and sequencing

The moltv53A locus was mapped using F2 offspring of a
Tübingen×WIK reference cross
(Rauch et al., 1997) with a
panel of simple sequence length polymorphism (SSLP) markers
(Knapik et al., 1996) on pools
of 48 mutants and 48 wild-type siblings and localised to linkage group 17.
Linkages were confirmed and refined by genotyping single mutant and sibling
embryos. To confirm that foxa2 is tightly linked to the
moltv53A mutation, a polymorphism within the
foxa2 gene was scored by cleavage with the enzyme Tsp509 I.
To identify the lesion in foxa2, RNA pools from 20
mol-/- and 20 wild-type embryos at 24 hpf were reverse
transcribed. The entire coding sequence of the foxa2 gene was
amplified by PCR using the forward and reverse primers
TTCCAGGATGCTCGGTGCTGTCAAAATGG and GTCACAAGGTCCAAGAGAGTTTAGGAAG. The product
was cloned into the TOPO TA vector (Invitrogen) for sequence analysis.
Automated fluorescent sequencing was carried out using an ABI 373A sequencer.
Internal foxa2 primers were designed and 17
moltv53A and seven wild-type samples analysed. Sequence
analysis was also carried out on PCR products from single embryo genomic DNA
from five moltv53A, three wild-type and nine siblings. The
molst20 locus was mapped with a panel of SSLP markers
(Knapik et al., 1996) using
progeny obtained from crosses of F2 founder fish heterozygous for
molst20. Initial mapping of the
molst20 locus to LG 17 was performed on mutant and
wild-type DNA pools obtained from 20 embryos per pool. Linkages were confirmed
and refined by genotyping single mutant and wild-type sibling embryos. The
mutation was identified by amplifying and sequencing foxa2 exons with
template DNA prepared from tail fins of heterozygous
molst20 fish. The part of the second exon containing the
mutation was amplified and sequenced from the 5′ and 3′ direction
using the primers CAGCACA-CCCTGACATTTCTTT and GTGATTGAACGAGTAGTGATGTT,
respectively. The presence of the mutation was confirmed in 108 single
molst20 embryos by PCR with the primers
TACCATGA-GCCCAATGGCAG and CGAGTGGCGGATAGAGTTT, and subsequent cleavage of the
PCR product with the restriction enzyme MseI.

Transplantation experiments

Mosaic analysis was carried out between wild-type and mutant embryos at
different stages. Biotin (1%)-injected donor cells were taken from dome stage
embryos and placed into mol-/- or wild-type hosts at the
shield stage. Cells were placed in the shield, some of the cells of which are
destined to form floorplate (Woo et al.,
1995). Embryos were left to develop until 32 hours of development
and were then fixed for analysis. At this time point, it was possible to score
genotype of donor and host embryos by morphology. Embryos were stained for
foxa gene expression by in situ hybridisation and position of
transplanted cells was determined by revealing the biotin using the Vectastain
Elite kit. In some cases, embryos from these and other experiments were
embedded into JB4 medium (Polysciences) and sectioned at 10 μm for further
analysis.

Results

monorail encodes foxa2

We identified monorail (moltv53a) as a locus
important for patterning of axons that cross the midline in the midbrain and
hindbrain through screening existing lines of fish for mutations affecting
midline axon guidance (Fig.
1F,G; data not shown). mol-/- embryos were
originally identified by their curled down tail and lethality by 5 days
post-fertilisation (Fig. 1A,B)
(Brand et al., 1996). A second
allele (molst20) was isolated in a screen for mutations
affecting myelin basic protein (mbp) expression, and this
allele shows a similar phenotype (Fig.
1C).

To elucidate the role of the mol gene product, we cloned the gene
affected by the mutations. Mapping and sequence analysis revealed linkage to,
and mutations within, the conserved forkhead box of foxa2 in both
alleles (Fig. 1E; see Fig. S1
in the supplementary material). Furthermore, injection of foxa2
morpholino antisense oligonucleotides phenocopies the curly tail phenotype of
mol-/- embryos (Fig.
1D). To confirm that the defects in mol-/-
embryos are due to a loss of Foxa2 function, we attempted to rescue the
phenotype by expressing functional Foxa2 in mutant embryos. Embryos were
injected with RNA encoding a 17β-estradiol inducible Foxa2-ER construct
(Rastegar et al., 2002). To
assess phenotypic rescue, we focused upon the organisation of the facial motor
nucleus (CNVII), which is fused at the midline in mol-/-
embryos (Fig. 1F,G). Activation
of the Foxa2-ER fusion protein during gastrulation rescued this phenotype in
48% of mutant embryos (Fig.
1H). Altogether, these data allow us to conclude that the gene
mutated in mol-/- embryos is foxa2.

mol-/- embryos possess a floorplate that lacks
lateral cells

Previous studies have suggested that Foxa2 mediates floorplate development
downstream of Hh signalling in amniotes
(Sasaki and Hogan, 1994) and
downstream of Nodal signalling in fish
(Rastegar et al., 2002). To
investigate if this is indeed the case, we assessed floorplate induction and
differentiation in mol-/- embryos. To our surprise,
slightly enlarged cells with typical cuboidal floorplate morphology
(Odenthal et al., 2000;
Hatta et al., 1991) were
evident along the entire axis of the CNS in moltv53a
embryos (Fig. 2A,B). We
therefore conclude that Foxa2 is not essential for induction of a
floorplate.

The floorplate is reduced in width in both mol-/- and
smu-/- embryos. (A-C) Lateral views of the floorplate in
the trunk of living wild-type, mol-/- and
smu-/- embryos. Cuboidal floorplate cells are present in
all embryos, although the cells are increased in size in
mol-/-. (D-F) Dorsal views of foxa expression in
the hindbrain floorplate. In the mol-/- and
smu-/- mutants, expression is reduced from a six cell wide
band to a one to three cell wide band (black lines). Additionally, gaps of
expression are present in the mol-/- mutant. (G-I) Dorsal
views of foxa2 expression in the head. Midline expression is narrow
in both the mol-/- and the smu-/-
mutant, and additionally is patchy in the midbrain and hindbrain in the
mol-/- mutant. e, endoderm; fp, floorplate; hy, hypochord;
nc, notochord; ZLI, zona limitans intrathalamica.

Subsequent to its initial induction during gastrulation, the floorplate
laterally expands such that it eventually encompasses MFP and LFP cell
populations. We next assessed whether the floorplate of
mol-/- embryos expands laterally. In wild-type embryos,
both foxa and foxa2 are expressed in the hindbrain in a band
of cells six or seven cell diameters wide that encompasses the MFP and LFP
(Fig. 2D,G). Expression of both
genes is reduced to a one to three cell wide row in mol-/-
embryos (Fig. 2E,H). From this,
we conclude that the floorplate in mol-/- embryos lacks
laterally positioned cells. This could be due to loss of MFP and/or loss of
LFP components of the floorplate. The narrow floorplate phenotype is similar
to smu-/- embryos that lack zygotic Hh signalling and
possess MFP but not LFP cells (Chen et
al., 2001; Varga et al.,
2001) (Fig.
2C,F,I). However, unlike smu-/- mutants,
mol-/- embryos show gaps of foxa and
foxa2 expression at stereotypic positions in the posterior midbrain
and hindbrain, and reduced levels of expression of these genes in the spinal
cord (Fig. 2E,H and see
below).

Expression of regulatory genes is not maintained in the floorplate of
mol-/- embryos

Expression of regulatory genes expressed in the MFP of wild-type embryos is
not maintained in mol-/- mutants. Dorsal (A-E,J,K) and
lateral (F-I) views of wild-type (D,F,H,J) and mol-/-
embryos (E,G,I,K), at ages shown in the bottom left-hand corner, analysed for
expression of various genes (indicated in the bottom right-hand corner). (A-C)
Clutches of embryos from parents heterozygous for the
moltv53a mutation. No obvious differences in twhh,
her9 or ntn1 expression in prospective floorplate between
wild-type and mol-/- mutants is evident at these early
stages. The arrowheads in D,E show gaps in floorplate shh expression
in the mol-/- embryo compared with wild type.

Although initial steps of floorplate development occur in
mol-/- embryos, gaps soon start to appear in the ventral
midbrain and hindbrain expression domains of shh and other floorplate
markers (Fig. 3D,E; data not
shown). At later developmental stages, all early markers of floorplate
analysed, including foxa1, shh and twhh, show reduced and
patchy expression in the hindbrain and reduced or absent expression further
caudally (Fig. 3F-K).
Expression of these genes in the forebrain showed no major changes in
mol-/- embryos (data not shown). TUNEL analysis of
mol-/- embryos did not reveal any increase in apoptosis of
floorplate cells, suggesting that midline cells were still present even though
gene expression is reduced (data not shown). These results indicate that Foxa2
is required for the maintenance of expression of regulatory genes in the
floorplate.

Development of floorplate in mol-/- embryos does
not depend on Hh activity

We next performed experiments to assess whether mol-/-
floorplate shares features of wild-type MFP or LFP. Hh signalling is essential
for induction of LFP but is not required for development of MFP
(Odenthal et al., 2000;
Schauerte et al., 1998).
Therefore, if the floorplate remaining in mol-/- embryos
is LFP, abrogating Hh activity should abolish its development. To reduce Hh
activity, we treated wild-type and mol-/- embryos with the
Hh pathway inhibitor cyclopamine
(Incardona et al., 1998). In
the trunk, mol-/- embryos still possessed a
morphologically distinct floorplate following cyclopamine treatment
(Fig. 4B).

Floorplate develops in mol-/- embryos independently of
Hh activity. Lateral views (A,B), dorsal views (C-H) and transverse sections
(I-R) of wild-type (A,C,E,G,I,K,M,O,Q) and mol-/-
(B,D,F,H,J,L,N,P,R) embryos with (A,B,D,G,H) and without (C,E,F,I-R)
cyclopamine treatment. (A,B) Both the wild-type and the
mol-/- embryo treated with cyclopamine have cuboidally
shaped floorplate cells (arrowhead) at 20 hpf. (C,D) Clutches of embryos from
parents heterozygous for the moltv53a mutation. Treatment
with cyclopamine between the 4-hour and 2-somite stages did not appear to
affect expression of twhh in either wild-type or
mol-/- mutants. (E-H) Wild-type and
mol-/- embryos showing patchy ntn1 expression
after treatment with cyclopamine. (I-R) Transverse sections through the spinal
cord of wild-type and mol-/- embryos showing changes in
floorplate expression of various genes in the mutants. cpm, cyclopamine.

Early markers of floorplate such as twhh
(Ekker et al., 1995),
ntn1 (Strähle et al.,
1997b) and her9
(Latimer at al., 2005) are
predominantly or exclusively expressed in the MFP of wild-type embryos. The
initial retention of expression of these markers in mol-/-
embryos (Fig. 3A-C) suggests
that at early stages, the floorplate of mol-/- embryos is
similar to the MFP of wild-type embryos. Furthermore, expression of twhh,
ntn1 and other early floorplate markers is retained in
mol-/- (and wild-type) embryos following cyclopamine
treatment (Fig. 4C-H; data not
shown). Unlike other floorplate markers, ntn1 expression is retained
in the spinal cord midline at later stages in mol-/-
embryos, albeit at reduced levels.

The observations that a floorplate still forms in
mol-/- embryos with compromised Hh signalling and, at
early stages, expresses markers restricted to the MFP of wild-type embryos
indicates that the floorplate of mol-/- embryos shares, at
least at early stages, some features of the MFP of wild-type embryos.

The absence of markers that clearly and exclusively label the LFP of
wild-type embryos made it more difficult for us to assess if the residual
floorplate of mol-/- embryos shares features with
wild-type LFP. Markers of both MFP and LFP in wild-type embryos, such as
foxa, are expressed in a single row of midline cells in the trunk
spinal cord of mol-/- embryos
(Fig. 4K,L). Nkx2.2 genes may
initially be expressed throughout the ventral most spinal cord but over time,
expression is lost in midline floorplate cells. Both nk2.2a
(Barth and Wilson, 1995) and
nkx2.2b (Schäfer et al.,
2004) are expressed in a narrower band of cells in the spinal cord
of mol-/- embryos compared with wild-type embryos
(Fig. 4M-R). The absence of
midline expression evident in wild-type embryos was less apparent in
mol-/- embryos (Fig.
4O-R). This suggests that at late somite stages, the residual
floorplate in mol-/- embryos expresses markers that would
be localised to LFP in wild-type embryos. Together, these observations suggest
that at late somite stages, the expression profile of the residual floorplate
of mol-/- embryos matches neither MFP nor LFP of wild-type
embryos.

Floorplate fails to differentiate in mol-/-
embryos

The results described above show that although medially positioned
floorplate cells with typical cuboidal morphology are present in
mol-/- embryos, these cells fail to maintain the
expression of several key regulatory signals and transcription factors. We
next asked what the consequences of these defects are upon the differentiated
character of the floorplate. Mature medial floorplate expresses the
extracellular matrix protein Col2a1 (Lele
and Krone, 1997), the secreted proteins Mindin1 and Mindin2 (Min1,
Min2) (Higashijima et al.,
1997), Connective tissue growth factor (Ctgf)
(Dickmeis et al., 2004), and
the homeodomain protein Arx (Miura et al.,
1997).

Expression of all these markers of floorplate differentiation is severely
reduced or absent in the spinal cords of mol-/- embryos
and reduced/patchy further rostrally (Fig.
5B,E,H,K,N; data not shown). Other sites of expression, such as
notochord or hypochord, are not obviously affected by the
mol-/- mutation. This failure in floorplate
differentiation is in striking contrast to the situation in
smu-/- embryos, which maintain MFP cells
(Chen et al., 2001;
Varga et al., 2001). In
smu-/- embryos, expression of all medial floorplate
differentiation markers is very similar to wild type
(Fig. 5C,F,I,L,O). As virtually
all Hh signalling is absent in smu-/- embryos, these data
indicate that Foxa2 mediates floorplate differentiation independent of the Hh
pathway.

Floorplate differentiation fails in mol-/- but not in
smu-/- embryos. Lateral (A-I) and dorsal (J-O) views of
the trunk floorplate in 36 hpf (A-I) and 24 hpf (J-O) wild-type,
mol-/- and smu-/- embryos. (A-O)
Expression of floorplate differentiation markers col2a1, min1, min2,
arx and ctgf is similar in wild-type and
smu-/- embryos but expression is severely reduced or
absent in the mol-/- embryos (despite the presence of
floorplate cells, arrowhead in B). Although floorplate expression is retained,
the somitic expression of arx and ctgf is lost in the
smu-/- mutants (L,O). fp, floorplate; hy, hypochord; nc,
notochord.

The floorplate defects in mol-/- embryos are not
only due to reduced levels of Hh activity

Given that Hh signalling is not required for expression of MFP
differentiation markers, the loss of such markers in
mol-/- embryos is unlikely to be a consequence of the
failure to maintain Hh gene expression in the floorplate. However, as Hh
signals are required for LFP formation, then reduced Hh activity in
mol-/- embryos could contribute to the loss of lateral
cells in the mol-/- floorplate. To address this issue, we
overexpressed shh in mol-/- embryos and assessed
consequences upon floorplate-specific gene expression.

Injection of shh RNA leads to ectopic dorsal expansion of the
MFP/LFP marker foxa in the midbrain of both wild-type and
mol-/- embryos (Fig.
6A,B) (Schauerte et al.,
1998; Odenthal et al.,
2000). However, in mol-/- embryos, exogenous
Shh failed to restore the normal width of the floorplate
(Fig. 6B). This suggests that
Foxa2 is required downstream of Shh to promote the expression of
foxa. As expected, shh injections had no effect upon the
differentiated MFP marker col2a1 in either wild-type or
mol-/- embryos
(Odenthal et al., 2000) (data
not shown).

Floorplate phenotypes in mol-/- embryos are not only
due to reduced Hh activity. (A,B) Lateral views of wild-type and
mol-/- embryos following overexpression of exogenous
shh. There is some dorsal expansion (arrowheads) of foxa
expression in the midbrain of both the wild-type and the
mol-/- embryo; however, foxa expression remains
discontinuous and narrow along the CNS midline of the
mol-/- embryo (asterisks in B). (C,D) Transverse sections
of embryos in which wild-type cells were transplanted into the prospective
floorplate of a mol-/- embryo (C) or
mol-/- cells were transplanted into the prospective
floorplate of a wild-type embryo (D). foxa is shown in blue and
transplanted cells are stained brown.

To address if the reduced levels of Hh activity do contribute to the
floorplate defects in mol-/- embryos, we transplanted
wild-type cells into the prospective floorplate of mol-/-
hosts (n=9). In some cases, wild-type cells expressed the floorplate
marker foxa (not shown), whereas in others they did not
(Fig. 6C). As wild-type cells
should possess all the proteins required for floorplate differentiation, the
most likely interpretation of this result is that the
mol-/- environment fails to provide cell non-autonomous
signals (such as Hh proteins) to either induce or maintain floorplate marker
expression in the wild-type cells. Further supporting this conclusion,
mol-/- cells transplanted into a wild-type environment
(n=9) often maintained expression of foxa in lateral regions
of the floorplate (Fig. 6D).
This indicates that there is not an absolute requirement for Foxa2 in the
maintenance of foxa expression and that non-autonomous signals
arising from a wild-type environment can alleviate the
mol-/- floorplate phenotype.

Altogether, these results provide evidence that Foxa2 functions both
upstream of Hh signalling (in the regulation of Hh gene expression) and
downstream of Hh activity (in the induction/maintenance of expression of
various floorplate markers).

ntn1 and nk2.2a are ectopically expressed in the
midbrain and hindbrain of mol-/- embryos

Consistent with other markers of floorplate maturation, reduced levels of
expression of ntn1 are retained in the spinal cord floorplate of
mol-/- embryos (Fig.
4F). However, with this marker, we found a more complex phenotype
in the midbrain and hindbrain. Transcripts are absent from the most ventral
CNS cells. However, ectopic patches of expression are observed in ventricular
zone cells positioned more dorsally (Fig.
7B,E). The extent of mis-expression varied from a few cells to
large clusters of cells. Similarly, analysis of the homeobox gene
nk2.2a showed that like ntn1, ventricular zone expression in
the hindbrain is more dorsally positioned in mol-/-
embryos than in wild type (Fig.
7H). Unlike other mol-/- phenotypes, the
dorsal expansion of nk2.2a and ntn1 expression shows highly
variable expressivity.

As nk2.2a expression is dependent on Hh signals (e.g.
Barth and Wilson, 1995;
Varga et al., 2001), we
wondered whether the ectopic dorsal expression of ntn1 and
nk2.2a depends upon Hh signals acting through transcription factors
other than Foxa2. smu-/- embryos show no ectopic dorsal
expression of either ntn1 or nk2.2a
(Fig. 7C,F,I); however, the
ectopic expression may be dependent upon loss of Foxa2 activity. We therefore
analysed mol-/-;syu-/- embryos that have
reduced Hh activity. In these embryos, the ectopic dorsal expression of
ntn1 and nk2.2a was considerably reduced
(Fig. 7M,N). Similarly,
mol-/- embryos treated with cyclopamine between 36 and 48
hpf show no ectopic ntn1 and nk2.2a expression
(Fig. 7O; data not shown).
Together, these results suggest that Hh activity is required for the ectopic
expression of ntn1 and nk2.2a in mol-/-
embryos. However, we never observed ectopic expression of any Fox genes
(foxa, foxa1, foxa2, Fig.
7K and data not shown) or indeed Hh genes (data not shown) in a
pattern similar to that of ectopic ntn1 or nk2.2a
expression. Altogether, these results indicate that in the absence of Foxa2
function, Hh activity dependent ectopic expression of ntn1 and
nk2.2a occurs via unknown transcription factors.

Foxa2 is required for formation of the trochlear nuclei and for
bilateral separation of the facial and oculomotor nuclei

In order to analyse the induction and patterning of neurones adjacent to
the floorplate in mol-/- embryos, we crossed a GFP
transgene driven by isl1 regulatory elements into the
mol-/- line (Fig.
8B). This transgene labels cranial motoneurones from soon after
their birth (Higashijima et al.,
2000).

A prominent phenotype in mol-/- embryos is that the
facial (cranial motor nucleus VII) and oculomotor (III) neurones form single
nuclei at the midline rather than bilateral pairs of nuclei
(Fig. 8C,D). Although the
number of facial motoneurones is not obviously reduced in
mol-/- embryos, the oculomotor nucleus has fewer neurones
than in wild type (Fig. 8E,F).
There is a similar reduction in posterior trigeminal motoneurones (Vp) and the
trochlear nucleus (IV) is completely absent in mol-/-
embryos (Fig. 8E,F), which
consequently lack a trochlear decussation
(Fig. 8G,H). Whereas the facial
nucleus is positioned at the midline in mol-/- embryos,
more rostral trigeminal motoneurones almost always remain as bilateral nuclei.
One potential reason for this is the consistent retention of expression of
other Fox family genes in floorplate cells between the two trigeminal motor
nuclei (Fig. 8I,J).
Complementing the changes in positioning of neurones, axons in the medial
longitudinal fasciculi were closer together or occasionally fused in the
region of the facial nucleus but remained normally positioned lateral to the
floorplate at all other positions (data not shown). These results indicate
that Foxa2 activity within the floorplate is required for the induction of
specific cranial motor nuclei and for the mediolateral positioning of other
motoneurones and axon pathways.

Foxa2 is required for formation of the serotonergic raphé
nucleus

The hindbrain raphé is the major location of serotonergic neurones
in the brain. Because these neurones differentiate close to the floorplate
(Ye et al., 1998;
Pattyn et al., 2003), we
examined their development and differentiation in mol-/-
embryos. To do this, we analysed expression of a tryptophan hydroxylase
encoding gene (tphR; tph2 - Zebrafish Information Network)
(Teraoka et al., 2004) that
encodes a key enzyme in the synthetic pathway for serotonin production.

By 3 days of development in wild-type embryos, serotonergic raphé
neurones are positioned in anterior and posterior clusters that probably
correspond to the dorsal and ventral subdivisions of the mature nucleus
(Fig. 9A,C)
(Bellipanni et al., 2002). By
contrast, mol-/- embryos are virtually devoid of
tphR-expressing cells with the few remaining neurones located
caudally within the raphé (Fig.
9B,D). tyrosine hydroxylase (th)-expressing
neurones in the diencephalon and other catecholaminergic neurones all appear
to differentiate as normal in mol-/- embryos
(Fig. 9E-H).

At least two possibilities could explain the loss of serotonergic neurones
in mol-/- embryos. The first is that there is an intrinsic
requirement for Foxa2 in serotonergic neurones or their precursors.
Alternatively, the failure in differentiation of floorplate tissue may
secondarily lead to a failure in production of signals such as Shh, important
for induction or differentiation of raphé neurones
(Ye et al., 1998;
Teraoka et al., 2004).
Supporting the possibility that loss of Hh signalling activity leads to loss
of raphé neurones in mol-/- embryos, expression of
exogenous shh restored tphR expression
(Fig. 9O,P; 43% of mutant
embryos). This result suggests that foxa2 is not autonomously
required in these neurones, and that their absence is due to reduced Hh
signalling.

mol-/- embryos show defective oligodendrogenesis
in the midbrain and hindbrain

In a screen for zebrafish mutants that show myelination defects (H.-M.P.,
B.D. and W.S.T., unpublished), a second allele of mol-/-
(molst20) was isolated. Olig2 is a basic helix-loop-helix
transcription factor expressed in precursors of both motoneurones and
oligodendrocytes (Park et al.,
2002) and myelin basic protein (mbp) and
proteolipid protein (plp) both mark myelinating cells
(Brösamle and Halpern,
2002). By 36 hours, there is a reduction of olig2
expression in the hindbrain (Fig.
9I,J) and during later development, there is a reduction of both
mbp (Fig. 9K,L) and
plp (Fig. 9M,N) in the
midbrain and hindbrain, suggesting a loss of myelin forming cells in these
areas.

Discussion

In this study, we have characterised the zebrafish
mol-/- mutant phenotype and show that mol encodes
foxa2. A floorplate is induced in mol-/- embryos
but fails to maintain expression of various Fox family transcription factors
and Hh signals. Subsequently, the floorplate fails to expand laterally and
fails to differentiate. Surprisingly, despite the severity of these defects,
cells with floorplate morphology remain viable through the first few days of
development. In addition to the role in floorplate development, Mol/Foxa2 is
required for differentiation and patterning of various ventral CNS cells,
including serotonergic raphé neurones, cranial motoneurones and
oligodendrocytes. Our results suggest that defects in these cells in
mol-/- embryos are probably due at least in part to
regulation of Hh and possibly other signals by Foxa2. Curiously, we observe
ectopic activation of Hh target genes in dorsally positioned ventricular cells
of the midbrain and hindbrain. This phenotype suggests that Foxa2 may help
limit the range of activity of Hh proteins. Altogether, our data allow us to
propose a revised model for midline signalling and floorplate development in
zebrafish (Fig. 10).

Summary of proposed role for Foxa2, Nodal and Hh signalling pathways in
ventral CNS development. The model is based upon data in this paper and in
other papers cited in the text. Nodal signalling induces MFP through Madh/Smad
and FoxH1/Fast/Sur transcription factors. This occurs in the absence of Foxa2
function, implicating other factors downstream of Nodal in the earliest
induction of floorplate identity. Nodal signalling induces foxa2
expression in MFP and Foxa2 function is required for differentiation of these
cells. Hh genes are among those requiring Foxa2 function for maintained
expression. However, as Hh genes are initially expressed in
mol-/- embryos, we suggest that other transcription
factors, including Madh2/FoxH1, contribute to the presence of Hh proteins in
the ventral neural tube. The only known role for Hh proteins in the MFP is to
maintain expression of differentiation markers. Hh signals working through Gli
proteins induce foxa2 expression in the LFP. Foxa2 activity is
required for proper formation of the LFP and this is likely to be due both to
non-autonomous roles (e.g. regulation of Hh production in the MFP), and
activity within the LFP itself. Hh activity also spreads further to induce and
pattern adjacent ventral CNS cell types, including cranial motoneurones and
serotonergic raphé neurones. In the absence of Foxa2 function, Hh
activity leads to ectopic dorsal expression of several Hh pathway target
genes. This implies that Foxa2 may also negatively regulate the dorsal spread
of Hh activity within the ventral CNS.

Foxa2 is not required for floorplate induction in zebrafish

Our analysis of mol-/- embryos shows that floorplate is
induced at all axial levels in the absence of normal Foxa2 function. Even in
the midbrain and hindbrain, where expression of floorplate markers is severely
reduced, floorplate cells are still recognisable by their cuboidal morphology.
Furthermore, the medial longitudinal fasciculi are displaced towards the
midline in mutant embryos but rarely fuse, as they do in Nodal mutants,
suggesting that at least some of the signalling properties of the floorplate
cells are retained. From these observations, we conclude that initial
floorplate induction in zebrafish can occur independently of Foxa2
function.

If zebrafish Foxa2 mediates floorplate expansion and differentiation (see
below), then what might be the identity of the transcription factors required
for floorplate induction? Other Foxa family genes are clear candidates, but
morpholino knock-down of foxa or foxa1 alone produces no
obvious phenotype (Rastegar et al.,
2002). Alternatively, there could be cooperation and redundancy
between related Fox family members. Although our data do not address this
issue with respect to floorplate induction, there is very little redundancy at
later stages. Foxa2 is required to maintain expression of other Foxa genes and
so all Foxa activity is severely compromised following loss of Foxa2 activity
alone.

In zebrafish, floorplate induction initially requires Nodal signalling.
Transcription factors directly downstream of this signalling cascade such as
Madh2/Smad2 and Foxh1/Sur/Fast1 induce floorplate markers cell autonomously
most probably by directly binding to the promoter/enhancer elements of
floorplate-specific genes (Müller et
al., 2002). However, the widespread requirement for these
components of the Nodal pathway in the development of other cell types (e.g.
Kunwar et al., 2003) indicates
that, alone, they are unlikely to confer specificity to the induction of
floorplate markers. Other transcription factor encoding genes may play a role
in the initial steps in induction of the floorplate, including her9
(Latimer et al., 2005),
tbx (Amacher et al.,
2002) and homeobox (Jeong and
Epstein, 2003) genes. Indeed, given the complexity of the
regulatory regions of floorplate-specific genes (discussed in
Strähle et al., 2004),
cooperation between many of these proteins may be required to induce
floorplate identity.

In contrast to mice lacking Foxa2 activity
(Ang and Rossant, 1994;
Weinstein et al., 1994;
Hallonet et al., 2002)
mol-/- embryos do not appear to have any major mesodermal,
endodermal or anterior CNS defects. This may reflect significant differences
in the developmental cascades used in mice and fish. Alternatively, it may be
due to different Fox family genes mediating similar events in different
species. Zebrafish frequently have additional homologues of mammalian genes,
most probably owing to a genome duplication event in the lineage leading to
teleosts (Postlethwait et al.,
1998; Woods et al.,
2000). When such duplicated genes are retained, the original roles
of the ancestral gene are predicted, in some cases, to be divided between the
duplicates (Force et al.,
1999). Although we do not know if this is the reason for the
difference in phenotypes between mice and fish lacking Foxa2 function, the
expression of other Foxa genes in mesendodermal derivatives in fish (Odenthal
et al., 1998) is consistent with the possibility that such genes play an
equivalent role to Foxa2 in mice.

Foxa2 is required for floorplate differentiation

Although cuboidally shaped cells with typical floorplate morphology are
present in mol-/- embryos, they fail to maintain
expression of Foxa and Hh family genes and lack expression of markers normally
restricted to the differentiated MFP of wild-type embryos. Our favoured
interpretation of this phenotype is that Foxa2 regulates floorplate
differentiation. The requirement of Foxa2 for maintained expression of
floorplate regulatory genes is unequivocal. However, our interpretation of a
requirement for Foxa2 during floorplate differentiation is based upon the
assumption that the floorplate of mol-/- embryos shares
identity with the MFP of wild-type embryos (and that loss of MFP
differentiation markers therefore reflects a failure in floorplate
differentiation). If instead, Foxa2 mediates induction of MFP, then the
absence of MFP differentiation markers would reflect the absence of the MFP
rather than a failure in differentiation.

There are two lines of evidence that make us favour the idea that Foxa2
mediates floorplate differentiation rather than MFP induction. The first is
the absence of severe floorplate defects in mol-/- embryos
until mid-somite stages (for example, the MFP markers twhh, her9 and
ntn1 are expressed as in wild type). This suggests that a structure
equivalent to the MFP of wild-type embryos is initially present in
mol-/- embryos. Second, disrupting Hh activity in
mol-/- embryos fails to completely abolish the floorplate
that remains. As LFP induction in zebrafish is reliant on Hh pathway
signalling (Odenthal et al.,
2000; Etheridge et al.,
2001), then if the residual floorplate in
mol-/- embryos was equivalent to LFP of wild-type embryos,
we would expect that abrogating Hh activity should result in complete loss of
floorplate identity. As this does not happen, then we think that the
mol-/- floorplate is more similar to the Nodal
pathway-induced MFP than the LFP of wild-type embryos. Together, these
observations support the conclusion that floorplate induction occurs in
mol-/- embryos but differentiation fails.

Although we suggest that the floorplate in mol-/-
derives from MFP precursors, it does appear to abnormally express markers
normally restricted to LFP. nk2.2a and nkx2.2b have both
been considered as LFP markers as their expression is excluded from MFP
(Strähle et al., 2004;
Schäfer et al., 2004) and
both genes show some patchy and/or reduced expression in the ventral midline
spinal cord of mol-/- embryos. Similarly, in late stage
cyc-/- mutant embryos, floorplate tissue expresses a
combination of MFP and LFP markers (Albert
et al., 2003). Ventral CNS Nk2 genes are regulated between
thresholds of Hh activity (e.g. Jacob and
Briscoe, 2003) and the altered spatial expression of
nk2.2a and nkx2.2b in mol-/- embryos may
reflect the lowered levels of Hh activity in the mutants. Taken together, our
results suggest that the floorplate in mol-/- embryos is
derived from MFP, but exhibits some gene expression characteristics of the LFP
of wild-type embryos.

The mechanisms by which Foxa2 regulates floorplate differentiation are
likely to be both directly through binding to the regulatory regions of
floorplate differentiation genes and indirectly through the transcriptional
control of regulatory genes (e.g. Chang et
al., 1997; Müller et al.,
1999; Rastegar et al.,
2002; Epstein et al.,
1999). For example, the reduction of foxa2 expression in
mol-/- mutants suggests that positive autoregulation by
Foxa2 activity is required to maintain foxa2 expression. Analysis of
the expression of other class 1 Fox family members (foxa, foxa1)
revealed a similar dependence on Foxa2 activity and so all these transcription
factors, and probably others, may mediate Foxa2-dependent floorplate
differentiation. Although Foxa2 regulates expression of Hh genes, these are
unlikely to play a significant role in MFP differentiation as this occurs
normally in embryos with severely reduced Hh signalling
(Etheridge et al., 2001;
Chen et al., 2001;
Varga et al., 2001).

Foxa2 functions in the lateral expansion of the floorplate

Although a floorplate forms in mol-/- mutants, it never
expands laterally to acquire the full width of floorplate tissue of wild-type
embryos. The lateral expansion of the floorplate is a hh-dependent
process in zebrafish (Odenthal et al.,
2000) and probably in chick
(Charrier et al., 2002) and
other vertebrates. Various studies have suggested that Foxa2 (and/or highly
related genes) functions in a positive regulatory loop with Hh genes, whereby
Hh gene activity induces foxa2 expression and Foxa2 activity promotes
hh expression (reviewed by
Strähle et al., 2004).
This model was originally proposed to explain how Hh signals from the
notochord could induce foxa2 in the floorplate that would in turn
induce hh expression in floorplate cells. In zebrafish, the initial
induction of foxa2 in the prospective floorplate is dependent upon
Nodal, rather than Hh, activity (discussed in
Strähle et al., 2004),
but as we discuss below, the regulatory loop between Hh and Foxa2 proteins may
contribute to the failure of lateral expansion of floorplate tissue in
mol-/- embryos.

The absence of lateral cells with floorplate identity in
mol-/- suggests that Foxa2 usually has a role in both MFP
and in LFP precursors. We suggest that Nodal signals initially induce Foxa2
expression but in the absence of Foxa2 activity, there is a progressive loss
of Hh expression in the floorplate. As a consequence, the reduced levels of Hh
activity in the MFP may be insufficient to induce (or maintain) LFP identity.
However, the absence of laterally positioned floorplate cells in
mol-/- embryos is unlikely to be due solely to reduced Hh
activity within the floorplate. For example, at early stages, transcription of
hh genes appears to be normal in mol-/- embryos.
Indeed, LFP cells can still form in cyc mutant embryos that lack MFP,
suggesting that Hh signals from other sources are sufficient to induce LFP
(e.g. Albert et al., 2003).
Furthermore, overexpression of shh fails to restore the full width of
the floorplate in mol-/- mutant embryos suggesting that
Foxa2 function is required within LFP cells for these cells to differentiate
with floorplate identity.

Foxa2 activity may limit the range of Hh activity

Within the midbrain and hindbrain, loss of Foxa2 activity leads to
reduction of ntn1 and nk2.2a expression in ventral CNS
cells, but surprisingly there is variable ectopic activation of both genes in
dorsal cells close to the ventricle. This implies that the normal activity of
Foxa2 is required to limit the activation of these genes to cells at, or close
to, the midline. Both nk2.2a (e.g.
Barth and Wilson, 1995;
Varga et al., 2001) and
ntn1 (e.g. Müller et al.,
2000) are regulated by Hh activity and so we predict that the
ectopic expression of these genes is due to ectopic activation of the Hh
signalling pathway. This idea is supported by the observation that abrogation
of Shh activity in mol-/- embryos reduces the ectopic
nk2.2a and ntn1 expression. Although a dependence on Hh
signalling is evident, all our other analyses suggest that the level of Hh
signalling is considerably reduced in the ventral CNS. How, then, could
reduced levels of Hh activity ventrally lead to ectopic Hh signalling
dorsally?

Hh signalling can negatively regulate Hh target genes through the induction
of genes that limit the range/efficiency of Hh signalling. For example, the
transmembrane protein Patched is induced by Hh signalling and appears to
sequester Hh protein, thereby limiting its range of action. This was elegantly
demonstrated by showing that Hh signals spread more efficiently across clones
of cells expressing a modified form of Patched that fails to bind Hh proteins
(Briscoe et al., 2001). Other
proteins such as the EXT family members, Tout-velu, Brother of tout-velu and
Sister of tout-velu also regulate the range of activity of Hh signals
(Han et al., 2004;
Takei et al., 2004). Therefore
one possibility is that Foxa2 is required to induce proteins that subsequently
limit the range of Hh activity (as expected, patched expression is
severely reduced in mol-/- embryos, data not shown).
Although this is an attractive possibility, the source of Hh proteins that
lead to ectopic activation of nk2.2a and ntn1 is not
obvious. There is very little hh transcription in floorplate cells of
older mol-/- embryos, and so one possibility is that Hh
proteins may come from underlying tissues such as the notochord or even gut
endoderm as has been proposed in other situations
(Wijgerde et al., 2002). An
alternative possibility is that Hh proteins come from other cells in the CNS.
hh expression is unaffected in the diencephalon and anterior midbrain
of mol-/- embryos. Although these hh expressing
cells are some distance from the site of ectopic expression, the third
ventricle provides a route by which secreted Hh proteins could potentially
move along the AP axis of the CNS. Indeed, the ectopic expression of
nk2.2a and ntn1 is tightly restricted to cells adjacent to
the ventricles.

mol-/- embryos exhibit a variety of defects in
ventrally positioned cells adjacent to the floorplate, including a severe
reduction in the serotonergic raphé neurones and prospective
oligodendrocytes. There are two possible mechanisms by which Foxa2 could be
involved in the development of these cell types. Specification may be due
either to a direct requirement for Foxa2 within the precursors of the cell
groups or, alternatively/additionally, Foxa2 may regulate signals that
influence cell specification.

Overexpression of exogenous Shh restores some tphR-expressing
prospective raphé neurones in mol-/- embryos,
implying that there probably is not a cell-autonomous requirement for Foxa2 in
the specification of these neurones. Hh signalling is required for
specification of raphé neurones (Ye
et al., 1998; Teraoko et al., 2004) and so the simplest
explanation of the serotonergic neurone phenotype of
mol-/- embryos is that the reduced levels of Hh activity
lead to a failure in induction of this cell type. It is perhaps surprising
that injection of shh RNA can rescue the raphé neurones given
the short lifetime of the injected RNA and the relatively late appearance of
serotonergic neurones. However, it is currently unknown when serotonergic
neurone precursors are first specified, nor is it known at what stage in the
specification/differentiation of these cells that Hh signalling is
required.

The reduction of expression of mbp, olig2 and plp
expression in the hindbrain of mol-/- embryos provides the
first evidence that Foxa2 is required for oligodendrocyte development and
consequently myelin formation. It is not known if the oligodendrocyte
precursors express Foxa2 and we have been unable to determine if the loss of
these cells in mol-/- embryos is due to reduced Hh
activity (data not shown). Further work will be required to determine how
Foxa2 mediates the development of the oligodendrocyte lineage.

Unlike prospective oligodendrocytes and serotonergic neurones, most cranial
motoneurones are specified normally in mol-/- embryos.
This suggests that Foxa2 is not essential for specification of most
motoneurones and that levels of Hh activity are still sufficiently high in
mol-/- embryos at the stages at which the motoneurones are
induced. The anterior motor nuclei, however, are reduced or absent in
mol-/- mutants. We have not resolved whether there is a
cell-autonomous requirement for Foxa2 in the specification of these cells.

A revised model of floorplate formation in zebrafish

Taken together, our analyses of the mol-/- mutant and
other studies allow us to revise existing models of floorplate formation in
zebrafish (Fig. 10). Nodal
signalling induces MFP (and floorplate-specific genes such as shh and
foxa2) through Madh/Smad and FoxH1/Fast1/Sur transcription factors.
This occurs in the absence of Foxa2 function, implicating other transcription
factors, perhaps including Her9 (Latimer
et al., 2005), downstream of Nodal in the earliest induction of
floorplate identity. Downstream of Nodal activity, Foxa2 function is required
for maintained expression of Fox and Hh family genes and for differentiation
of the floorplate. Hh signals produced in the MFP subsequently contribute to
the induction of foxa2 (and foxa) expression in more lateral
cells. Foxa2 activity is required for lateral expansion of the floorplate,
probably owing both to non-autonomous roles (e.g. regulation of Hh production
in the MFP) and to activity within the LFP itself. Hh signalling also spreads
further dorsally to induce and pattern adjacent ventral CNS cell types,
including cranial motoneurones, serotonergic raphé neurones and
oligodendrocytes. In the absence of Foxa2 function, Hh activity leads to
ectopic dorsal expression of several Hh pathway target genes, implying that
Foxa2 also negatively regulates Hh activity within the ventral CNS by an
unknown mechanism.

Acknowledgments

We thank Matthias Schäfer and other colleagues for reagents, Jon
Clarke and members of our groups for discussions, and Marika Kapsimali,
Juliette Mathieu, Gillian Brunt and Filippo Del Bene for comments on the
manuscript. This work was supported by grants from the Wellcome Trust to
S.W.W. and M.R.; from the BBSRC, Royal Society and EC to S.W.W.; from the
German Human Genome project to R.G.; from the Japanese Ministry of Education,
Science, Sports and Culture to H.T.; from the Deutsche Forschungsgemeinschaft
by stipend DFG P807/1-1 to H.-M.P.; and from the National Multiple Sclerosis
Society and Wadsworth Foundation to W.S.T. S.W.W. was a Wellcome Trust Senior
Research Fellow.

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